CA1222704A - Magnetic particles as supports for organic synthesis - Google Patents

Abstract

ABSTRACT
A support system for organic synthesis comprising magnetic particles in a dispersion medium covalently attached to functional groups having affinity for polymer subunits, and method for making and using the support system, e.g. for synthesis of oligodeoxynucleotides and polypeptides.

Description

~Z2~7~L

I`he present invention relates to compositions, products and methods for use in synthesis of organic compounds, more specifically to pxoducts and methods relating to the use of rnagnetic particles covalently bound to reactive moieties as supports for organic synthesis.
Chernical transforrnations requiring the interaction of two or more species, reagents or substrates, often require the separation of product from excess reagent, untransformed reactants, by-products, solvents, etc., after the reaction is 10 completed. Such separations are costly, tirne consuming, and often lirnit the quality and purity of the product produced.
To avoid these problerns, chernical transformations have been conducted in media containing two phases, with one participant in the reaction affixed to a solid phase, ~hile the rernaining cc~nponents reside in solution. The solid phase is often an organic polymeric substance, hence the terms "solid-phase reactions" and "poly~ner supported reactions" are often used synonymously. The art of solid phase synthesis has expanded rapidly in recent years, and has been reviewed in: P. Hodge and D. C. Sherrington, editors, Polymer-supported Reactions in Orc~anic Synthesis, N.Y., John Wiley and Sons, 1980.
The ease of operation of reactions and other advantages have led to the use of solid supports for executing nurnerous chemical transformations, e.g., in the synthesis of polypeptides -- on polystyrene beads, B. Gutte, et al., J. Biol. Chem., 246: 1922 (1971), and synthesis of DN~, RNA or polypeptides on marcoporous silica gel , S. L. Beaucage et al ., Tet. Letters 22:1859 (1981);
Matteucci and Caruthers, J. Am. Chern. Soc. 103(11):3185-91 (1981). However, previous methods of chemical transformation 30 that employ reagents or substrates on solid supports suffer from some serious disadvantages.

,~

- 2 - 1 2 2 2 7 0 ~
Because of the porous nature of the supports con-ventionally used for synthesis of DNA, RNA, polypeptides, poly-mers or other oligomers, for e~ample, most of the grc~ing oligcnlers are attached to walls on the inside of the solid support. This gives rise to a problem of accessibility bet~een the grc~ing oligc~mer and the reagent. Large reagents are not accessible to the grcwing nucleic acid, and are generally excluded altogether. Other reagents for elongation and chemical transfonmation must diffuse slch~ly through the support to react with the supported substrate.
Thus, molecules in solution entering into chemical reaction with molecules affixed to the support must diffuse through the pores to react. These disadvantages lead to slow reaction rates, often not the same for all supported molecules, incomplete reactions, and poor yields, especially when the diffusing molecule is large. Further, different classes of sites (e.g. within or without the pores) have displayed different kinetic behavior and synthetic failure.
The absence of unifonn reagent accessibility has also adversely affected attempts at oligomer synthesis via automation, and other problems have occurred as well. For example, automation has proven difficult to apply to chemical synthesis because channels form in the beds of conventional solid supports through which reagents must pass. This channeling effect is not con-ducive to the fonnation of hcmogeneous product. The hetero-yenities which result lead to synthetic oligcmers which are difficult to purify - the desired oligc~er (is produced) is hard to separate from contaminating oligomers which do not have the desired structure.
~ synthesis totally in solution is preferable and provides cleaner products and higher net yields. See H. Yaj~na and N. Fujii, J. Chem. Soc. (Per]sins Trans I) :789-831 (1981).
Ho~ever, synthesis in solution requires the tedious isolation of each synthetic intermediate product.

3 :~LZ227~
-It has now been found that non-porous magnetic particles can provide excellent supports for synthesis of organic compounds, especially for oligomers such as DNA, RNA, polypeptides, polymers and other multi-unit molecules having defined sequences, by attaching a grwwing oligomer chain to a small magentic particle.
These supports permit chemical transformation having all of the advantages of previous solid supports listed above, yet none of the disadvantages of such supports.
Reaction work-up is simplified because the supported species are easily separated frcm the non-supported species by magnetic removal. In some cases, this makes it possible to avoid exposing the reaction product(s) to water or to avoid a chromatographic separation in the reaction. If an excess of a reagent results in a greater reaction yield, then the excess can be used without causing separation problems. When a magentic particle-supported reagent is used, the spent reagent is easily recovered and can possibly be recycled. This is very important economically, ~nd can make it worthwhile to prepare complex supported reagents. Since, in most cases, magnetic separation and washing of the oligomer are all that is required to work up the reaction product at any particular stage, it becomes feasible to automate the process. As magnetic particles are insoluble and non-volatile, they are non-toxic and odorless. Hence, carrying out reactions involving toxic and odiferous compounds affixed to - the solid support may be more acceptable environmentally than the corresponding reactions in solution.
As the separation of magnetic particle-supported products from excess reagents and by-products is uncomplicated and easily accomplished, it is possible to perform a sequence of reactions repetitively on a single substrate bound to a solid support. This is valuable for synthesizing oligomeric substances such as DNA, R~A, and polypeptides with defined sequences of nucleic acids or amino acids, by repetitively adding a single nucleotide or amino ~ ~ i , ~

~2~ 0~

acid to a grc~7ing chain of such monomer units covalently affixed to the solid support.
Preferably the magnetic support particles are single domain magnets, es~ist as colloidal suspension in the reaction mi~ture, and are "superparamagnetic" exhibiting no residual ferromagnetism. C. P. Beam and T. D. Livingston, J. Appl. Phys., Suppl. to Vo. 30:120 (1959). Preferably the particles are magnetite particles, although they can also be other magnetic metal or metal oxides, whether in pure, alloy or composite form, so long as they have a reaetive surface. Other materials that may be used individually or in combination with iron, include but are not limited to cobalt, nickel, silicon, etc. Methods of making magnetite or metal or metal oxide particles are disclosed in Vandenberghe et al, "Preparation and Magnetic Properties of Ultra-Fine Cobalt Ferrites," J. of Magnetism and Magnetic Materials 15-18:1117-18 (1980); E. Matijevic, "Monodispersed Metal (Hydrous) Oxides - A Fascinating Field of Colloidal Science" Acc.
Chem. Res. 14:22-29 (1981).
Previous utilization of magnetic particles has included:
magnetie fluids in the blood, R. Ne~7bower, IEEE Transactions on Magnetics MAG-9, ~45 (1973); attaehment of functional groups for separation of biomoleeules, U.S. Patents 3,970,518 to I. Giaver;
labeling of cell-surface receptors, S. Margel et al., J. I~m.
Meth. 28:341-53 (1979); attachment to drugs for magnetic target-ing during therapeutics, A. Senyei et al., J. App. Phys., 49(6):
3578 (1978), K. Widder et al., Pro. Soe. Exp. Bio. Med., 58:141 (1978), K. Mosbaeh and U. Sehroeder, FE2S Letters 102:112 (1979);
seleetive separation of viruses, baeteria and other cells, R.
Molday et al~, Nature 268:438 (1977); and incorporation of magnetie partieles as support in gel affinity chromatography for biological polymers, K. Mosbach and L. Anderson, Nature 270:359 (1977). Hc~7ever, such particles have not previously been used as supports for chemical synthesis.

-t ~, ~2227~

The partieles in accordance with the invention have a specific functionality covalently bound to their surface by a coupling agent. The magnetite particles are derivatized by a silylation, perrnitting selective support of desired functional groups. Growing oligomers may then be linked to the appropriate funetional groups, permitting the synthesis of DNA, RNA, polypeptides or other oligcmers using the same chemistry for oligomer elongation that is used on elassieal solid supports.
Preferably, the remaining reactive groups on the surface of the magnetic particles are blocked frorn further reaction, e.g. with non-reactive silane, prior to carrying out the oligomer synthesis.
The preferred magnetie particles are non-porous, which permits all attached reagents and substrates to be affixed to the surfaee of the particle. Bloekiny reactive sites on the partieular surfaee which are not reaeted with the eoupling agent prevents unwanted side reaetions. Furtherrnore, such derivatized magnetic particles preferably exist as colloidal suspensions. As such, reagents and substrates affi~ed to the surface o~ the particle extend direetly into the solution surrounding the particle. They react with dissolved reagents and substrates in solution with rates and yields characteristie of reaetions in solution, rather than rates assoeiated with previous solid supported reactions.
Colloidal suspensions of the magnetic particles are diffieult or impossible to remove frcm solvent by filtration.
However, they are readily separated from solution by application of a rnagnetic field.
Additional advantages acerue if the particles are srnall, preferably less than 100,000 Angstroms. With decreasing size, the ratio of surfaee area to volume of the partieles increases, permitting rnore funetional groups to be attaehed per unit weight of magnetie partieles. Furt~ermore, the smaller the partiele, the - 6 - ~2~2~
better it stays in colloidal suspension. Finally, additional advantages accrue if the diameter of the magnetic particles is less than approY.imately 10,000 Angstr~ns. Preferably, the magnetic particles are single domain magnets, which display superparamagnetism, resulting in stronger attraction in eY~ternal gradients of magnetic fields, and an absence of residual ferromagnetism after the external field is removed.
The ~gnetic particles can be used as solid supports in the same capacities as those now occupied by previous solid supports. This includes attaching small molecules to the support that are able to undergo chemical transformation and easy recovery, or alternatively, attaching reagents to the solid support, pe~l~itting the easy separation of the product in solution and/or the recovery and recycling of the reagent.
I~le coupling or linking agent must be able to attach to the particle and must be readily reactable with the desired oligomer subunit, or with other linker subunits which can be readily reacted with the desired oligottler subunit to form an appropriate support therefore. In that way, one end of the ~0 coupling agent is covalently bound to the particulate colloid, in suspension, and the oligonler subunit, in turn, is bound, preferably covalently, to the other end of the coupling agent.
Further oligomer subunits are brought in contact with the oligcmer subunit attached to the support ("initial olig~ner subunit") so that a series of oligomer subunits can react with each other sequentially, thus forming an oligomer which is bound to the initial oligomer subunit.
The preferred bonding or linking agents in accordance with the present invention possess appropriate reactivity at each end of the molecule, for the magnetic support molecules and the initial oligcmer subunit, respectively. The preferred coupling agents for use with this invention are silane lin]sing agents, which comprise a silicon portion, which has reactivity with oxygen or hydro~yl groups on the metal particle surface, and an organic _ 7 _ ~ ~2%7~4 portion, which provides an easily reactable functional group, e.g., amino, carboxyl, hydroxyl, etc., so that oligomer subunits can be readily attached at the end of the molecule. The inorganic reactive end of the molecule is tailored to the metal, metal oxide or other inorganic material which will serve as the synthesis support. The organic functional end of the bonding or coupling agent should be structured to react with a subunit or precursor of the specific oligomer to be synthesized. A number of preferred types of organosilane lin]cing agents are disclosed in Sili_on Compounds, Register and Review, published by Petrarch Systems, Bristol, Pa., (1982), e.g. trialkylsilylchlorides and diallcylsilyldichlorides.
This invention provides all of the advantages of previous solid supports for oligomer synthesis without the accompanying disadvantages. Deoxyribonucleic acid oligomers ten bases long have been routinely prepared in greater than 95% yield for each coupling step using magnetic solid supports. This contrasts markedly with similar synthesis on silica supports, e.g.
silica gel, where yields are erratic and occasionally as low as 10~ for a single coupling step.
While not wishing to be bound by theory, it is believed that the superior results achieved with the present invention may arise, at least in part, from the fact that the sites of attachment of the grcwing oligcmer are distributed on the surface o the particle, which itself is in a colloidal suspension, and thus are uniformly available to successive reagents dissolved in solution. With the preferred non-porous particles, functional groups are not contained in restrictive pores, and reagents need not diffuse through pores to reach the sites for reaction. Since the particles are in suspension, not in a bed, channeling does not occur as a result of successive reagent treatments. The improved separability of the supported oligomer synthesis sites may also contribute to the improved results.

1.~`

270~

It is an object of the present invention to provide a solid support for the chemical transformation of reactive molecules affixed to that support, which displays all of the advantages of classical solid supports but none of the disadvan-tages. Another object of the present invention is to provide a solid support system for the chemical transformation of reactive molecules affixed to a support uniformly available to reagents in solution and readily separable from those reagents.
It is also an object to provide a non-porous particle to which functional groups may be attached and still not be confined to restrictive pores. It is also an object of this invention to provide a particle effectively in solution to which functional groups may be attached as precursors for a molecular elongation process. It is also an object of the present invention to pro-vide a method of polymer synthesis adaptable to automation pro-ducing a relatively high yield. It is also an object to provide a process for separation of synthesized oligcmers from a support system utilizing magnetic particles.
It is also an object of this invention to provide a method for the synthesis of oligomers such as DNA, RNA, and polypeptides that is adaptable to automation producing relatively high yields.
q'he invention herein comprises compositions, products and m2thods for an in vitro support system for synthesis of organic oligomers, and separation of the oligomers fro~ the reactants. Magnetic non-porous particles of small dimension (preferably 10-100,000 Angstroms) are covalently bound to functional groups, such as anuino, carbox~1, hydro~1, etc., via silylation.
Remaining nucleophilic sites are preferably blocked by silylation.
A growing polymer chain is then attached to the functional group.
The particles preferably exist as a colloidal suspension in a dispersion rnedium. The magnetic particle acts as a solid supnort system for synthesis of high molecular weight substances and the ~;r ~227~)4L
g col]oidal suspension all~s the reaction to occur as effectively as if the re~ctants were in solution. l'he product may be isolated and subsequently removed by activation of a magnetie field to collect the support system.
In aecordance with the present invention, small particles of magnetie metal or metal oxide are attaehed to an organosilane eoupling agent, ~hieh in turn is attaehable to an oligcmer subunit so that oligomer subunits ean be attaehed to the support system and used for olig~ner synthesis. For example, magnetite [Fe304]
partieles ean be prepared by pyrolysis of ferrous formate partieles in a stream of dry argon at 350C. The particles of ferrous formate may be prepared by drying an aerosol of a ferrous formate solution in a stream of heated air at 180-220C. The average size of the magnetite particles can be controlled by varying the concentration of the ferrous formate solution, by varying the size of the aerosol droplets, or by milling, as will be appreeiated by those s]cilled in that art.
m ese magnetie particles may preferably then be coated with silica, e.g., by reaetion with solutions of sodium silieate in water or in mixtures of ethanol and water.
l'he compositions of the present invention are made-by derivatizing the support partiele by silylation. Silylation is the replaeement of an aetive hydrogen of a protie material with a substituted silicon atom. Preferably, the derivatized support particle is then brought in contact with reagents containing oligomer subunits under reaction conditions whieh will vary depending upon the type of oligomer being constructed and the funetional group of the eoupling agent.
The preferred coupling agents for use in the present invention are organosilanes of the general formula:
si(x)n[Rm(y)p]4-n (I) lX2%7~)4 where Si is the silicon oE the organosilane coupling or lin]cing agent, X is a leaving group i.e., an organic moiety which can be displaced by the bonds forrned between silicon and the reactive groups on the support surface. X may be alkox~y, preferably lower alkoxy, alkenyloxy, al]caryloxy, aryloxy, al]cynyloxy, halo or aTnino, preferably a secondary aTnino e.g., dialkylarnino. R is a linking group - a bond or an organic rnoiety which can link the silicon, or another lin]cing group attached directly or indirectly to the silicon, to a functional group which can serve as the point of attachment of the oligorner subunit which is to be bound to the substrate, e.g., the initial subunit of the olig~ner which is to be synthesized. Because it is a linker, R must be at least bifunctional, but R may also be tri- or tetrafunctional. Thus, any given R group may be bound to up to three functional groups of further lin]cing groups, in addition to the moiety through which it is directly or indirectly attached to the silicon. Typically, R
will be al]cylene, e.s., methylene or polymethylene, and where it is desired to space the functional group(s) which will ultimately take part in the synthesis reaction in a position which is removed frorn the metallic support, R rray be long chain alkylene, e.g., preferably a long chain polysnethylene. However, R may generally be any multifunctional derivative of al]cyl, alkylene, alkenyl, alkynyl, aryl, al]caryl or aralkyl groups, and can include ester, aTrino, amido, ether, thioether or other linking functional groups where the group consists preferably of more than five atoms in length and more preferably fifteen atoms or ~ore. Y is simply a functional group which can react with and bind the reactant to its desired support, e.g., an oligorner subunit, to bind that reactant, through the lin]cing groups and silicon bonds, to the synthesis support particle. The nature of the functional group depends on the nature of the reactant, (e.g. initial oligorner subunit) to which it is to bind. Preferably, Y is an amino, hydroxyl, carboxyl or other functional group ~lhich will covalently attach to 22;~7~A~
a linker group and which will covalently combine with the reactant it is desired to support (e.g. an olig~ner subunit). "n" is an integer having a value of 1-3, reflecting the fact that the silicon can be attached to up to three leaving groups, in addition to the linker chain for the initial oligomer subunit. At least one of those leaving groups must be replaced with a bond between the silicon ,~nd the particle surface, and up to three may be so replaced. "m" is an integer having a value of at least 1, which simply reflects the fact that there must be at least one linker to bind the initial oligonler subunit to the silicon portion of the coupling agent. Typically, one linker, R, e.g., a polymethylene unit of one to twenty carbons, will be used to support one functional group Y. However, Inore than one functional group can be attached to the organic portion of the coupling agent, either within the linker chain (e.g. Si-R-Y-R-) or branched from it, y (e.g.: Si-Ch2CH2CH-CH2-).
Preferably, m is 1-3; most preferably m is 1, having a value of at least 1. 1'his means that there must be at least one group attached to the silicon portion of the coupling agent with ~hich the initial oligomer subunit will bind. Preferably, p has a value of 1-3, most preferably 1. If there is more than one X, Y or R
group, each such group can be different fr~n the others. Many organosilane compounds of forsnula (I) are c nercially available, see e.g. Petrarch 5ysten~s, Inc., Silicon Compounds, ~E~.
In the particularly preferred coupling agents, X is l~er alkoxy or chloro, R is an alkylene group containing at least one amido functionality, preferably having a straight chain of at least five and preferably fifteen or more atoms, and Y is an amino, hydroxyl or carboxyl group. Such coupling agents include:
N-2-aminoethyl-3-aminopropyltrimethoxysilane Chlor~netllylphenyltrimethoxysilane N,N-dimethylaminopropyltrimethoxysilane

4[2-(trichlorosilyl)ethyl~pyridine , I

- 12 - ~2Z270~
3-Brcmopropyltrimethoxysilane, and l-Trimethoxysilyl-2-(p-m-amin~nethyl)-phenylethane, and the like.
Linkages between the coupling agent and the surface of the magnetic particle can be stabilized by covalently crosslinking some of the functional groups (Y) of the coupling agent having the general formula (I) to functional groups (Y) of other molecules of the same coupling agent or of other coupling agents having the general formula (I). Such covalent crosslinks can be chemically synthesized after the coupling agents are affixed to the surface of the magenetic particle either by direct linking between Y groups or by using bifunctional or polyfunctional crosslinking agents corresponding to the general formula:
[C]s I (Ia) ~[B]q~A~[B']r~
wherein B and B' are chemical bonds or functional groups which react to form covalent bonds with the functional group Y of the coupling agent and a linking group designated as A in equation la.
Linking group A is a chemical bond or functional group which bridges between the Y groups through the moieties B and B'. A may serve as the support for chemical synthesis, e.g. of amino acids or DNA or other olig ers by attachment of one of the elements of the oligomer (e.g. an amino acid for polypeptide synthesis or a nucleotide for DNA or DNA synthesis) to the A linking agent, either directly or through a reactive group designated as C in equation (la).
The letters q, r and s in equation l(a) represent integers, with q and r each preferably being at least l, and more preferably q and r amounts to 3 and 7. Where A contains a group which can form a direct attachment to the oligomer subunit of interest, s can be 0. Otherwise, the attachment can be formed via a reactive group(s) C where s is l, or greater. Preferably from s ~2~27~

is 0 or 2, and q and r is from 2 to 5.
Linking group A can be a chemical bond or any multi-functional derivative of alkyl, alklene, alkenyl, alkynyl and alkaryl, or aralkyl groups, and can have functionalities which include one or more of amino, amido, ether, thioether and others known in the art. Preferably A is a lcwer alkyl group, or a lower alkyl group containing an aryl, amino, amido, ether, ester, etc., functionality with lcwer alkyl meaning frcm one to about 4 carbon atcms.
B and B' may be lower alkyl, amino, carboxyl, hydroxyl, haloalkyl or other functional groups which react with the groups Y
in formula (I). B and Bl may be the same or may be different. C
may be a carboxyl, ester, amino, hydroxyl or other functional group which can form a bond or attachment with the oligomer subunit or other functional group which can form a bond or attachment with the oligomer subunit or other compound or chain to be supported by the magnetic particles and synthesized or otherwise reacted.
Examples of crosslinking agents of formula (Ia) includes the following:
~ ( 2)3 2 3 -OOC(CH2)3NHCO~ ( 2)3Coo This is a trifunctional crosslinking agent in which the follcwing elements can be considered in connection with formula (Ia):

A is ~ CONH(CH2)3--(CH2)3HNOC ~ CONH(CH2)3-B = Bl = COO -r ~ q = 2 c = -COOCH2CH3 ` -Page 1 4 ~222~4 s = 1 In that compound, the oligomer subunit would be bound to the unused carboxylic acid by de-esterification.
.

~CH2)3CCCC~2CH3 -Occ~cH2)2cc-~(cH2)2~-\ P2)2-~D~oO(C!~)2--COO
C:O (C~2 ) 2C~l}

.
This is a trifunctional crosslinking agent in which A = -N(CH2)2N-(CH2)-NH-B - Bl --CO(CH2)2COC~
C - (CH2)3 COOCH2CH3 q ~ r = 3, and 10 S- 1 . ~;
In this comFound, the oligomer subunit could be bound to the C
moiety at the carboxylate group by replacement of the ethyl ester group~ .
-ax~ (CH2 ) 2(~}NH (CE~2 ) 2-~ (CH2 ) 2NH (CE~2 ) 2~ (CH2 ) 2NE3CO (CH2 ) a)o -OOC(CH2)C~ -COC(CH2)2 CO
miS is a tetrafunctional crosslinking agent, in which:
A NH(CH2)2-N-(CH2)2-NH(CH2)2-N (CH2)2N
B - Bl = -C0-(CH~)2-COC-q + R = 4 s = O
In this embodiment, ~he oligomer sukunit to be worked on wauld be bound to the secondary amine of the linking group A e.g. by reacting a carboxylic acid derivative of the olismer sub~nit with the amine ~n the presence of a condensing agent, such as di~yclohexyl carbodiîmude ~DCC).
The crosslinking agents of the present invention can be prepared from available starting materi~ls and methods well kncwn in the art. For example, the first exeTplary cross linker may be prepared by condensed benzene 1,3,S-tricarboxylic acid with the ethyl ester of 4-amino butyric acid in the presence of ~CC, , .
. ` ' 'j' ' ~., .

- ]5 - ~2~27~
followed by limited hydrolysis. The second compound may be pre-pared by reacting ethyl[2-aminoethylene-2-aminoethylene-3-amino-butyrate] with succinic anhydride. The third crosslinker can be prepared by reacting tetraethylene pentaamine with four molar equivalents of succinic anhydride.
Alternatively, coupling agents may be covalently linked kefore they are attached to the surface of the magnetic particle using crosslinking agents having the general formula (la). Also, several organosilane compounds are cc~mercially available that contain two reactive silicon coupling agents of the general formula (I) connected by crosslinking group, see e.g. Petrarch Systems, Inc., Silicon Compounds, supra.
Coupling agents of this type include:
bis[3-(trimethoxysilyl)-propyl]ethylenediamine.
bis[3-(triethoxysilyl)propyl]amine.
bis[3-(triethoxysilyl)propyl]tetrasulfide.
The most preferable coupling agents are ones obtained by reacting aminopropyltriethoxysilane and bis]3-(trimethoxysilyl)-propyl]ethylenediamine.
Using various silyl groups, particles may be produced having amino, carkoxyl, hydroxyl or other functional groups covalently attached to the surface of the particle. Procedures for the derivatization of metal oxides generally may be employed.
For example, reaction of magnetite particles suspended in dry toluene with coupling agents in accordance with formula (I) in which at least one Y is amino, such as aminopropyltriethoxysilane, yields particles covalently linked to an amino functionality. See O. R. Zaborsky, Meth. Enzymol., 44:317 (1976). It is preferred to avoid the use of solvents during silylation which may react with the organic end of the linking agents, such as amines or alkyl-thiols. I'he amino functionality can then be directly covalently attached to reagents or substrates to participate in a chemical .~

- 16 ~ ~2~27~
transformation, or longer spacer arms can be attached which, in turn, are covalently attaehed to the desired reagent or substrate.
For example, the amino functionality can be succinylated with succinic anhydride and reacted with an appropriately protected nucleoside in the usual manner for synthesis of DNA oligamers. See J. Am. Chem. Soc., 103, ~E~ Other reactions may require same rigorous conditions as will be appreciated by those skilled in the art.
As noted above, it is preferred that the magnetic particles be coated with silicon, e.g. fram an aqueous sodium silieate solution, prior to derivatization with the silyl group of choice. This results in a higher binding of the silyl groups being covalently attached to the particle surface. Without wishing to be bound by theory, it is believed that this occurs because this coating presents more of an opportunity for coupling agents to be bound and/or because the silicon oxide coating forms a strong intermediate bond to the ion in the particle, possibly by chemical reaction to form ion silicates, and/or the incamplete particles may be to same extent physically entrapped in a silicone oxide gel, to which the coupling of agents can be bound. The silicon coating is preferably formed in aqueous medium, e.g. by exposure of the particles to an aqueous silicate solution follawed by dehydration e.g. using non-aqueous solvents and/or heating, preferably in an inert atmosphere.
After the coupling agent has been attached to the magnetic particles, the remaining reactive groups on the particle surface are preferably blocked with blocking agents to prevent competition or interfering reactions. Known blocking agents can be used which are reactive with nucleophilic groups on the particle surface. Preferred are bloeking agents having the formula:
Si(X)n(R)4_n (2) ~2~ 4 where X is a leaving group, e.g., an alkoxy, halogen, amine, etc., preferably chloro; R is a group which is not a leaving group and which lacks any functionality which would cc~pete or interfere with the reactions needed to adjust the coupling agent, attach the initial oligomer subunit thereto, or carry on the oligcmer synthesis, such as alkyl, aryl, etc., and n is an integer having a value of 1 or 2. The R's can be the same or can be different. In the preferred case, X is chlorine, n is 1 and R is alkyl, preferably methyl.
During the blocking of unreacted sites on the particle surface, any Y's should be either a) unreactive with the blocking reagent or b) protected so as to be made unreactive with the blocking agent. In the preferred case, the unprotected functional group is carboxyl, which does not react significantly with trimethylsilylchloride.
When remaining reactive groups on the surface are blocked, e.g., trimethylsilylated, the particles are remarkably resistant to oxidation, reduction and acidic dissolution. Thus, the particles withstand 6N HCl, 0.2M iodine in water-tetrahydrofuran mixtures, and lM nitric acid for an extended period of time. The surface derivatization withstands all of the conditions required for chemical synthesis of DNA, RNA and peptides, including organic solvents (acetone, benzene, dimethylsulfoxide, nitromethane, tetrahydroEuran, ether, hexane, acetonitrile, methylene chloride, - chloroform, etc.), acids (zinc bramide, trichloroacetic acid, etc.), bases (pyridine, lutidine, 2 N NaOH, etc.), oxidants (iodine, nitric acid, etc.) and reductants (phosphites, sodium borohydride, etc.) The particles do appear to react with thiophenoxide. Thiophenoxide is a reagent often used to remove methyl groups fram methylphosphate esters which are intermediates of in vitro nucleic acid synthesis. See Example 1. Nucleic acid synthesized on magnetic oxide supports is most preferably removed with ammonium hydroxide fram the support prior to demethylation with thiophenoxide.

:,f~

- 18 - ~22~7~
Thus the support particle/linking agent oligc~er subunit syst~m of the present invention generally has the strueture indicated in formula 2a or 2b belcw:
M-O-Si(x)n[Rm(Y)p]3-n w (2a) wherein M is the support particle with the remaining nueleophilic sites blocked; O is the oxygen of the metal oxide or hydroxide, which is bound to the silicon; Si is the silicon of the organosilane linking or coupling agent; X may be as above-defined or may be replaced by additional covalent bonding to the surface of the support particle; n is an integer having a value of O, 1 or 2. R is as above defined; Y is as above defined; m is as above defined; p is as above defined.
Alternatively, if crosslinking agents are used, some of the functional groups Y of the support particle/linking agent oligomer subunit system of formula (3) are crosslinked to other Y
groups on the magentie par-tiele, in accordance with formula (2b):
M-O-si(X)n[Rm(Y)p]3-n[ ]q A-[C]sZn (2b) M-O~Si(X)n[Rm(y)p]3-n[B ]r wherein M is the support particle, O is the oxygen of the metal oxide or hydroxide, Si is silicon, X is a leaving group, R is a linking group, Y is a funetional group whieh can react with and bind the oligomer subunit or a group linked thereto, or cross linking agents B and B' which are chemical bonds or functional groups whieh react to form covalent bonds with the functional group Y, A is a linking group which bridges the Y groups and is bound to the oligomer subunit to be worked on, either direetly or ~hrough reactive group C, n is an integer having a value of 1 to 3, m is an integer having a value of at least 1, p is an integer having a value of 1-3, q and r are integers having the value of at least 1, q ~ r preferably being fr~n 3-7, and s is 0 to 5.
M in either formula is any magnetic particle, having - 19 - ~22270~

reactive groups on its surface, which can form bonds with silicon.
Preferably, the magnetic particles are less than 1 millimeter in average diameter, since the snaller the particle, the greater the surface area, and the more available the reactive groups attached to the surface t~ l be. More preferably, the particles are of a size which permits them to be contained in the reaction mixture in a colloidal suspension, e.g., belcw about 100,000 Angstrams. Most preferably, the particles are much s-naller, e.g., between about 10 and 10,000 Angstroms, and are single magnetic damain particles, which do not exhibit residual nagnetism when extracted from a magnetic field. Such single dcmain particles are "superparanagnetic", as discussed above. Such particles exhibit greater magnetic force in a given magnetic field per unit volume or per unit mass, than mu]tidamain particles.
The preferred material for component M is magnetite, although it can be other magnetic metals or metal oxides, whether in pure, alloy or composite form, so long as they have the required paramagnetism and reactive surfaces. Other materials that can be used in place of or in combination with iron include but are not limited to silicon, cobalt, nickel and other elements of Group VIII of the periodic table of the elenents. Such particles can be made by the methods disclosed in this application or by the methods discussed by Vandenberghe et al, or Matijevic, discussed supra, or may be purchased ccm~lercially from various sources, including Ferrofluidics Inc., of Nashua, New Hampshire, or the Bioclinical Group of Cambridge, Massachusetts.
In one embodiment, in accordance with formula (2a), M
is magnetite, Xn is (C2H50-)n, n is 1 or 2, R is N-trimethylene-carboxamido-dimethylene ( OE~-CH2-CH2-NHCO-CH2CH2), Y is a carboxyl group, m, w and p are 1, and Z is a deoxyribonucleic acid oligcmer subunit attached to Y. This represents the product of silylation of magnetite with addition of a second linker arm and an initial oligamer subunit. The reactions which produce such an embodiment may be written as follows, .~

- 20 - '~ ~2Z7~
with M representing the surface of the support particle:
M-OH + Si(X)n[Rm(Y)p]4_nM-O-si(x)n-l[ m p 3-n where n is an integer of the value 3, 2 or 1. In this reaction, at least one of the leaving groups (X) is replaced by the silicon oxygen bond.
pZ + M-O-Si(X)n l[Rm~Y)p]3_n M-o-Si~X)n_l [ m p 3-n Intermediate reactions may also take place, e.g. where the linking group ~R) is expanded by adding another linking unit, or where one functional group ~Y) is substituted for another.
Another preferred embcdiment is identical with the preceding one, except that M is magnetite coated with silica prior to derivatization with the particle surface.
Z Z
C C
M-o-si(CH2)3-N-(CH2)2N ~CH2)3 wherein O and M are as above defined; preferably M is magnetite, C is CO ~CH2)2 CO, and Z is a 2' deoxyribonucleotide oligomer subunit.
I'he method of use of appropriately derivatized particles in the present invention is direct. DNA can be synthesized in aqueous media using magnetic particles as supports. Preferably, the specific functionality covalently bound to the surface of the particle may be linked to the appropriate polymer and remain in solution pending completion of the reaction series and separation of the product. A standard repetition of a sequence of reactions, as described by S. L. Beaucage and M. A. Caruthers in Tetrahedron Letters, 22:1859-62 ~1981), produces oligomers of DNA attached to magnetic particles. This sequence will take approximately ten minutes per cycle and produces coupling in yields similar to the quanti-tative amounts mentioned above, as compared to synthesis on silica gel ~, - 21 - 1~227~4 supports where cycle times need to be greater than one hour to obtain optimum results.
The invention will be further understood with reference to the follcwing exan~les which are purely exemplary in nature and are not meant to be utilized to limit the scope of the invention.
Example Derivitization of Magnetic Particles Useful in DNA Synthesis A solution of barium formate was prepared by dissolving barium oxide pcwder in formic acid, the pH being adjusted to between 4 and 7 as a result. The concentration of the solution of barium formate was determined by precipitation of barium as its sulfate. This solution was then mixed with an equimolar amount of a freshly prepared solution of ferrous sulfate (0.05 M). The precipitate, barium sulfate, was removed by centrifugation. The supernatant contained a solution of ferrous formate having a concentration of approximately 0.04M.
This solution was diluted 10:1 with deionized, deaerated water, and then il~nediately passed through a Niro particle generator blo~ drier. In the blow drier, the solution was atomized to ~orm a fine aerosol, which was then dried in a stream of air between 190 and 210C, to produce a powder of finely divided ferrous formate, presumably accompanied by small amounts of ferric formate and hydroxide. The particles were then heated for three hours at 310C under an inert atmosphere (argon). The heating converted the particles of ferrous formate to particles of ferrous oxide, carbon monoxide, and water in accordance with the e~uation:
2 Fe(HCOO)2 = 2FeO + 2CO + H20 Evolution of carbon monoY.ide and water was detected during the course of the heating.
Ferrous o~ide is oxidized in ~nbient air. These particles, upon cooling, were exposed slcwly to atmospheric oxygen, during which exposure they were converted to magnetite (Fe3O4) according to the following equation:

- 22 ~ 27~4 3 FeO + 1/2 2 = Fe34 The particles were analyzed by o~cidation to ferric oxide (Fe2O3), and their size was determined by electron microscopy.
The particles used in this example had a median diameter of less than 500 Angstroms, and were single dc~ain, superparamagnetic particles. However, if the particles were not of the desired size, the process could be repeated with more concentrated solutions of ferrous formate for larger particles or more dilute solutions of ferrous formate for smaller particles. The particles can also be made smaller by milling them in a hia,h speed blender.
After the appropriate analytical procedures, the particles were derivatized. Finely divided magnetite (1 gram) was suspended in water (15 ml) with sonication. Aminopropyltriethoxy-silane (1 grarn) was then addecl to the aqueous mixture, and the pH was adjusted to ~ with 1 N HCl. The mixture was then stirred for one hour, after which the particles were recovered by application of a magnetic field. The particles were then washed and dried.
A spacer arrn was then attached to the amino functionality attached to the particles. rrhis was done by adding small portions of succinic anhydride to a stirred aqueous suspension of the particles. Several additions were made, and the pH was maintained at 7-8 throughout the addition by adding drops Qf 1 N NaOH. The product of the addition, magnetic particles to which were covalently appended the chain:
(C2H5)n SiCH2CH2CH2NHCCCH2CH2COOH, was collected by application of a magnetic field, washed with water and ethanol, and dried. The value of n will vary for particular rnagnetite/organosilane bonds, since the organosilane can react to forrn 1, 2 or 3 bonds with the reactive groups of the Inetal particles.
The dried particles were then suspended in anhydrous toluene and treated with trimethylsilylchloride. This treatment trimethylsilylated any unreacted nucleophilic sites, rendering . ~

-- 23 - ~222~4 them ~mreactive under conditions of subsequent synthesis as stated below. The particles were recovered with a magnetic field, washed with acetone and water, and used as supports for synthesis.

5'-O-Dimethoxytritylthymidine was attached by its 3'-hydroxyl group to the carbonyl group affixed to the magnetic particle, using dicyclohexylcarbodiimide as a condensing reagent, in a procedure similar to that used by Caruthers and c~orkers, Tet. Let., 22, supra. Successive elongation of the DNA chain was made by successive repetition of a sequence of three organic reactions. Each of the reacting reagents was added in an appropriate solvent, and the particles dispersed in the solvent to initiate the reaction. The reactions were as follcws:
1. Removal of the 5'-dimethoxytrityl blocking group, leaving a free 5'-hydrox~yl group, with a solution of zinc bromide in nitromethane, carried out at ro~m temperature, with occasional shaking. As the product of this reaction is brightly colored, the extent of the reaction could be detennined spectrophoto-metrically.
2. Condensation of 5'-dimethoxytritylthymidine-3'-O-dimethylaminometho~yphosphine with the free 5'-hydroxyl group of the thymidine, fram Step 1 above, attached to the magnetic particle through the Y carboxyl group, done in the presence of tetrazole as acid in acetonitrile as solvent. The reaction con-ditions were again room temperature, with stirring.
3.- Oxidation of the resulting trivalent phosphorus ccmpound formed above was accomplished by using a solution of 0.2 M iodine in a 1:2:1 water-tetrahydrofuran-lutidine mixture, at room temperature, with stirring.
These three steps are well knc~7n in the art as steps leading to the preparation of DNA oligomers. In this example, d(T10) was prepared, removed from the magnetic support with ammonium hydroxide, demethylated with thiophenoxide, and analvzed by sequence using the method of M~Lwam and Gilbert, Proc. Nat.

~, `~

- 24 - ~2~X~
Acad. Sci. U.S.A., 74:560 (1977).
The reaction sequences are as foll~s:
( 3 2 )3 ( 2 3 2 lAminopropyltriethoxysilane OH pH4 1 N HCl (OCH2CH3)2 MAGNETITE-O-Si(CH2)3NH2 + CH2CH2 OH pH7-8OC CO Succinic `o ~ Anhydride 1 N NaoH ~

( 2 3)2 MAGNETI~'E-O-si(CH2)3NHCOCH2CH2COOH + (CH3)3SiCl Trimethylsilyldlloride OH
Anhydrous Toluene - 25 ~ ~2227 (OCH2CH3)2 MAGNETI~-O-Si(CH2)3NHCOCH2CH2COOH
OSi(CH3)3 +

C6H5(MeOC6H4)2CO CH2 THYMINE

Dicyclohe~ryl- ¦ / O \
carbodiimide HC CH
\ CH-CH /
HO

5'-0-dimethoxy-tritylthymidine 6H5(MeOC6H4)2CO-C~2 THYMINE
HC CH
(I 2CH3)2 CH-CH2 MAGI~rITE-O-Si-(CH2)3NHCOCH2CH2COO

OSI(CH3)3 ZnBr2 CH3No2 Nitranethane ,.~

-Page 25~ .22;~:704 ~0C~2C~33) ~
MA~æTIT~Si (C~2 ) 3N~CH2CH2(OOH
bsi (CH3) 3 ¦ +
C6Els (Meoc6E34) 2C~CH2 rHyMIllE
Dl~clohexvl- . ~ ~ o car d~ e ` ICH-CH~
~10 5 '-0-dimet~
o~t~ityl-thym~ ne C6Hs (lleOC6H4) 2Ct}~ ~ _THYMINE
(OCH2(~H3)2 C CH-MA~NETITE-0-Si- (CH2) 3NHOOCH2CH2C00 OSi~ 3)31 ZnBr2 Nitrc~nethane 122270~

HCCH2 I~YMINE
~_- O
(oC11~CH3)2 CIH-~
~AG~'ITE-~5i(~ 2)3NHC~ 2CE12COO
CSi~3)3 C~13s~MeOC6H4)2CCX,~-h T~1INE
I HC -l. Tetrazole i Acetonitrile Cl~ OEk 2~ Iodine ~ ~k, Lutiuine, o-p~N~cH3)2 Tetrahydro~uran ~ \
5'-0-dimetho~ytritylthymidine-3'-C-dimethylaminomethoxyphosphine C6H5(Mecc6H4)2CC~CH2 0 rHyM~1E
H~ - CE~
C~CH2 I o O-P - CC~12 Tl-n~INE
OCH3~C- ~ OE~
( ~ 12CH3)2 CI~CH2 MAGNETITE~C-Si~CH2)3NHCOCE~CH
OSi(CH3)3 ,~
~se of Di~ferent Particles In a second example, particles available con~ercially as Biosorb C were obtained from the Bioclinical Group Inc. of Cambridge, ~ssachusetts. rrhe dimensions of these particles, were greater than lOOO but less than lOO,OOO Angstroms. These particles were of unknown composition, advertisecl only as having carboxyl groups covalently attached to magnetite. rrhe length and chemical rature of the spacer was ~nkncwn. m e particles were not single c30main, and were not superparamagnetic. ~et~7een each step, the particles were removed from suspension with a magnet, and then resuspended by dema~netization in the discharging field ~L2;~Z7~

of a demagnetizer.
5'C-dimethoxytritylthymudine was covalently coupled ~o the carboxyl group appended to the magnetic particles as described in steps 1, 2 and 3 in Example l, and an oliyomer ~en thymidines long was synthesized. The magnetic particles wer~-first reacted with trimethylsilyl chloride in anhydrous toluene, as described in Example l, so as to block any remaining nucleophilic sites on the surface of the particle. The oligomer was prepared in 70% yield based on the number of sites on the particle originally covalently bound to the monomer, the yield being judged by the extent of color released in the removal of the blocking 5'-0-dimethoxytrityl group in each cycler as measured at 400 nm in a spectrophotometer.

Silicon Coating of ~'~agnetic Particles Finely divided magnetite (2.27 gm.), prepared by the method described in Example lr was suspended in deionized water (30 mls~ r and a solution of sodium silicate (6 ml, Fischer, 40%) was added. The mixture was then sonicated (Branson sonic oscillator, microtip at power setting 3). T3 the resulting emulsion was added ethanol (95%r 7.3 ml) r and the mixture was shaken for 15 min.
reionized water was then added to the mixture of magnetic particles coated with silicate, and the particles were removed from suspension with a magnetr and repeatedly washed with hot water (30 ml) to re~ove excess sodium silicate until the washings .showed no formation of precipitate with addition of cupric sulfate solution. The particulates were then washed with cold water (2x 30 ml) and then twice with l:l nuxture of ethanol and water, and then with ethanolr then toluener and finally resuspended in 30 ml of anhydrous toluene. Excess water and ethanol was removed by azeotropic distillation with toluene.
Magnetic particles so prepared were then derivatized as described in EXample 1.

-Page 28~
27~L
Example 4 Crosslinked Functional Group Finely divided magnetite (2.0 ~3m.), prepared by the method descri~ed in Example 1, was suspended in dry toluene (25 ml) with sonicatiosl. To the suspension was added bis[3-~trimethoxysilyl)-prvpyl]-eth~lenediamine (2.0 ml of a 40%
solution in methanol), and the mixture was heated at reflux for 3 hours~ The Farticles were then removed frcm liquid by application of a magnetic field, ~ashed with acetone and methanol, dried, and then further derivatized as described in Example 1.
Additional advantages and modifications of the invention disclosed herein will occur to those persons skilled in the art.
Accordingly, the invention in its broader aspects is not limited to the specific details or illustrated example described.
m erefore, all departures made frcn the detail are deemed to be within the scope of the invention as defined by the apFended claims.

Claims (21)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Oligomer synthesis support comprising a magnetic particle covalently bound to oligomer sub-units by a cross-linked silane coupling or linking agent, said oligomer subunits comprising subunits of deoxyribonucleic acid or ribonucleic acid.

2. The composition of claim 1, wherein said oligomer subunits comprise subunits of deoxyribo-nucleic acid.

3. The composition of claim 1, wherein said magnetic particles comprise magnetite.

4. The composition of claim 1, wherein said magnetic particles are non-porous.

5. The composition of claim 1, wherein said oligomer synthesis support exists as a colloidal sus-pension in a dispersion medium.

6. The composition of claim 1, wherein said magnetic particles are single domain.

7. The composition-of claim 1, wherein the size of said magnetic particles is in the range of 10-100,000 Angstroms.

8. A method for synthesizing oligomers that comprises:
(a) supporting an initial oligomer sub-unit(s) on magnetic particles by a cross-linked silane coupling or linking agent, (b) reacting an oligomer subunit with said initial oligomer subunit, (c) and forming an oligomer product by sequentially repeating the attachment of oligomer subunits until the desired product is synthesized.

9. The method of claim 8, wherein the oligomer synthesis support particle comprises magnetite.

10. The method of claim 8, wherein said oligomer subunits comprise subunits of deoxyribo-nucleic acid, ribonucleic acid or polypeptides.

11. The method of claim 8, wherein said magnetic particles comprise magnetite.

12. The method of claim 8, wherein said magnetic particles are non-porous.

13. The method of claim 8, wherein said magnetic particles are single domain.

14. The method of claim 8, wherein said magnetic particles are in the range of 10-100,000 Angstroms.

15. The method of claim 8, wherein said magnetic particles are in the range of 10-100,000 Angstroms.

16. The method of claim 8, further comprising maintaining magnetic particles in a colloidal sus-pension in a dispersion medium during synthesis.

17. An oligomer synthesis kit having component materials capable of being reacted together to synthesize an oligomer comprising magnetic particles covalently bound to a silane functional group which is reactive to form a covalent bond with an oligomer subunit, and a source of oligomer subunits which are capable of chemically reacting with each other to form an oligomer.

18. The oligomer synthesis kit of claim 17, wherein there are a plurality of sources of oligomer subunits.

19. The oligomer synthesis kit of claim 18, wherein the plurality of sources of oligomer subunits comprise separate sources of deoxynucleotides con-taining the nucleosides deoxyadenosine, deoxy-guanosine, deoxycytidine and eoxythymidine.

20. The oligomer synthesis kit of claim 18, wherein the sources of oligomer subunits comprise separate sources of ribonucleotides containing the nucleosides adenosine, guanosine, cytidine and uridine.

21. The oligomer synthesis kit of claim 18, wherein the sources of oligomer subunits comprise separate sources of amino acids selected from the group of Glycine, L-Alanine, L-Valine, L-Leucine, L-Isoleucine, L-Serine, L-Threonine, L-Tyrosine, L-Phenylalanine, L-Tryptophan, L-Aspartic acid, L-Glutamic acid, L-Lysine, L-Arginine, L-Histidine, L-Asparagine, L-Glutamine, L-Cysteine, L-Methionine and L-Proline.